Diarrhea and experimentally-induced fluid secretion is almost always associated with HCO3-rich, plasma-like solution. In contrast, in vitro models rarely observe HCO3 secretion but have elegantly dissected the mechanisms of active Cl secretion for twenty-five years. A model of colonic HCO3 secretion is lacking. We have established methods to perform microperfusion of isolated rat and mice colonic crypts (the site for secretory processes) in which cyclic AMP-dependent and Ca-dependent agonists induce HCO3 secretion. Studies of cAMP-stimulated HCO3 secretion reveal a close association between HCO3 secretion and active Cl secretion with suggestion that apical HCO3 movement is via an anion channel, not C1-HCO3 exchange and that HCO3 is not generated endogenously, but from extracellular sources and its movement across the basolateral membrane (BLM) is a result of either the coupling of Na-K-2Cl co-transport (NKCCI) and Cl-HCO3 exchange or Na-HCO3 co-transport (NBC). We propose crypt microperfusion studies in normal rats and in CFTR knockout and NKCC1 knockout mice (and their control littermates) to establish the mechanism of HCO3 secretion. Initial studies with NKCCl knockout mice support the coupling of NKCC1 and Cl-HCO3 exchange as the mechanism of BLM uptake. Patch clamp studies will characterize the HCO3 conductance of anion channels in the crypt apical membrane. Although Cl movement across the basolateral membrane (other than that via NKCCl) has long been suspected, previous studies have failed to identify a carrier-mediated process, e.g., Cl-anion exchange. Using protease inhibitors to prepare BLM vesicles, we have recently identified a Cl-HCO3 exchange and plan experiments for its characterization. We believe AE2 encodes this Cl-HCO3 exchange and have cloned a full-length cDNA with 90 percent homology to gastric AE2 and propose its expression in HEK293 cells. We also have evidence that NBC in distal colon (i.e., cNBC) differs substantially from other NBCs and may represent a novel epithelial cell NBC. We propose comprehensive transport, electrophysiological and molecular studies to characterize crypt HCO3 secretion.